1. Field of the Invention
The present invention is related to the fields of molecular biology, virology, immunology and medicine. The invention provides a composition comprising an ordered and repetitive antigen or antigenic determinant array, and in particular an array comprising a protein or peptide of IL-5, IL-13 or eotaxin. More specifically, the invention provides a composition comprising a virus-like particle and at least one protein, or peptide of IL-5, IL-13 and/or eotaxin bound thereto. The invention also provides a process for producing the conjugates and the ordered and repetitive arrays, respectively. The compositions of the invention are useful in the production of vaccines for the treatment of allergic diseases with an eosinophilic component and as a pharmaccine to prevent or cure allergic diseases with an eosinophilic component and to efficiently induce immune responses, in particular antibody responses. Furthermore, the compositions of the invention are particularly useful to efficiently induce self-specific immune responses within the indicated context.
2. Related Art
A number of allergic diseases including asthma, nasal rhinitis, nasal polyps, eosinophilic syndromes and atopic dermatitis have prominent inflammatory components characterized by pronounced eosinophilic infiltration.
The most medically important group of these diseases, atopic asthma is recognized as a chronic inflammatory disease of the airways that is clinically characterized by episodic airflow obstruction, inflammation of the airways, and enhanced bronchial reactivity to nonspecific allergens. The degree of obstruction of the airways and hyperreactivity often correlates with the level of airway inflammation. These clinical features are indicative of asthma severity (Kay, A. B., J Allergy Clin Immunol, 1991, 87:893; De Monchy, J. G. et al., Am Rev Respir Dis, 1985, 131:373; Beasley, R. et al., Am Rev Respir Dis, 1989, 139:806; Azzawi, M. et al., Am Rev Respir Dis, 1990, 142:1407; Ohashi, Y. et al., Am Rev Respir Dis, 1992, 145:1469; Nakajima, H. et al., Am Rev Respir Dis, 1992, 146:374; Broide, D. H. et al., J Allergy Clin Immunol, 1991, 88:637; Warlaw, A. J. et al., Am Rev Respir Dis, 1988, 137:62). Cellular infiltration correlates with disease progression and indicates inflammation of the airways that is a major contributing factor to pathogenesis and pathobiology. The inflammatory infiltrate in asthma is complex; however, it is now widely recognized that CD4+ Th lymphocytes with a Th2 profile (Th2 cells) of cytokine expression play a pivotal role in the clinical expression and pathogenesis of this disorder (Robinson, D. S. et al., J Allergy Clin Immunol, 1993, 92:397; Walker, C. et al., J Allergy Clin Immunol, 1991, 88:935). Th2 cells regulate disease progression and airways hyperresponsiveness (AHR) by orchestrating allergic inflammation of the airways through the release of a range of cytokines such as IL-4, -5, -9, -10, -13 (Robinson, D. S. et al., N Eng J Med, 1992, 326:298; Robinson, D. S. et al., J Allergy Clin Immunol, 1993, 92:313; Walker, C. et al., Am Rev Respir Dis, 1992, 146:109; Drazen, J. M. et al., J Exp Med, 1996, 183:1). Like Th2 cells, the levels of eosinophils and their inflammatory products in the lung correlate with disease severity, and accumulation of this leukocyte in the airways is a central feature of bronchial dysfunction during the late-phase asthmatic response (Bousquet, J. et al., N Eng J Med, 1990, 323:1033). Although Th2 cells orchestrate many facets of the allergic response, their role in regulating eosinophilia through the secretion of IL-5 is thought to be a major proinflammatory pathway in asthma.
Interleukin-5 (IL-5) is a proinflammatory cytokine expressed at high levels in asthmatics. Moreover, IL-5 is a cytokine primarily involved in the pathogenesis of atopic diseases. It specifically controls the production, activation and localization of eosinophils, the major cause of tissue damage in atopic diseases. Furthermore, IL-5 is an inducible T-cell derived cytokine with remarkable specificity for the eosinophil lineage. IL-5 is controlled at the level of transcription and regulation of the gene represents a promising target for therapy of eosinophil-dependent allergic disorders such as asthma, eczema and rhinitis.
There is a large body of evidence that eosinophils are a key component of the allergic response in asthma. IL-5 is uniquely involved in the production of eosinophils, and with a variety of other cytokines such as IL-13, chemokines such as Eotaxin and other factors controls their activation, localization and survival. Thus, IL-5 has become an important drug target for new anti-asthmatics (Foster, P. S. et al., Pharmacol Ther, 2002, 94(3):253; Foster, P. S. et al., Trends Mol Med, 2002, 8(4): 162).
There is 71% homology between human and murine proteins (Cytokine hand book). IL-5 exhibits no significant amino acid sequence homology with other cytokines, except for short stretches in the murine interleukin-3, murine GM-CSF, and murine interferon-γ proteins. The predicted molecular mass of both the human and mouse protein sequences are 13.1 kDa. Biologically active IL-5 is a disulfide-linked homodimer that is covalently linked by highly conserved cysteine residues (44-86′ and 86-44′) that orient the monomers in a head to tail configuration (Takahashi T. et al Mol. Immunol. 27:911–920 1990). Although wild-type monomeric IL-5 is biologically inactive a functional IL-5 monomer has been engineered by insertional mutagenesis (Dickason R R, et al J. Mol. Med 74: 535–46 1996) Analysis of the crystal structure of human IL-5 demonstrated a novel two-domain configuration with each domain requiring the participation of two chains, with a high degree of similarity to the cytokine fold found in GM-CSF, interleukin-3, and interleukin-4 (Milburn M. V et al Nature 363: 172–176). The C-terminal region of IL-5 appears to be important for binding to the IL-5 receptor and for biological activity (Proudfoot et al J. Protein Chem. 15(5):491–9.1996). Binding of IL-5 to its receptor is thought occur in regions overlapping helices A and D where helix A is principally involved in binding the α-subunit of the receptor (Graber P. et al J. Biol Chem 270: 15762–15769 1995). Native human IL-5 has 2 potential glycosylation sites and mouse IL-5three. Human IL-5 is both N-glycosylated and O-glycosylated at Thr 3. Recombinant IL-5 expressed in eukaryotic systems exhibits a broad range of molecular masses from 45–60 kDa due to differential glycosylation. Deglycosylated IL-5 and IL-5 expressed in prokarytocic cells retain full biologic activity (Tominaga A. et al J. Immunol 144: 1345–1352, 1990).
The routes to drug discovery are typically based on screens for inhibitors of IL-5 production, ligand antagonists, control of receptor expression and receptor activation. In particular, inhibition of the action of IL-5 might provide a way of treatment against asthma and other diseases associated with eosinophils. Immunotherapy represents another and very attractive approach to controlling IL-5 levels and disease conditions associated with eosinophilia such as asthma.
Currently, the commonest treatment for prevention of the symptoms of asthma is the use of inhaled corticosteroids. Generally the use of these agents is fairly safe and cheap. However they function by inducing a general immunosuppressive effect and there are adverse side effects associated with their long term use including high blood pressure, osteoporosis and development of cataracts. Corticosteroids must be taken everyday and hence patient compliance is another issue in the successful use of these medicines. Furthermore there are asthmatic patients refractory to the use of corticosteroids necessitating the use of alternative therapies. Selective targeting of eosinophils using immunotherapeutic agents directed against IL-5 may overcome the adverse effects of using general immunosuppressive agents with pleiotropic actions.
Possible future treatment of diseases such as asthma may include passive immunization and, thus, the use of monoclonal antibodies specific for IL-5. Clinical trials with humanized monoclonal antibodies against IL-5 aimed at reducing eosinophilia in asthmatic patients are ongoing. In particular, clinical trials using SCH55700 (eslizumab, Schering Plough) which is a humanized monoclonal antibody with activity against IL-5 from various species [Egan, R. W. et al., Arzneimittel-Forschung, 1999, 49:779] and SB240563 (mepolizumab, Glaxo Smith Kline) which is a humanized antibody with specificity for human and primate interleukin-5 [Hart, T. K. et al., Am J Respir Crit Care Med, 1998, 157:A744; Zia-Amirhosseini, P. et al., J Pharmacol Exp Ther, 1999, 291:1060] have been reported. Both monoclonal antibodies demonstrated acceptable safety profiles in phase 1 trials and led to reduction of eosinophil numbers but no reduction in airway hypereactivity was, observed. The deleterious action that eosinophils exert on the airways of asthmatics is thought to be a chronic phenomena involving tissue re-modeling. Studies designed to test efficacy of anti IL-5 therapy in this context need to be assessed and are in development.
The treatment with mAbs, however, entails several disadvantages. Monoclonal antibodies are expensive therapeutic agents which must be taken monthly or bimonthly. The issue of patient non-compliance resulting form repeated medical visits for administration of the injected drug is an important problem. Furthermore, allotype variation between the patient and therapeutic antibody may lead to the monoclonal antibody therapy eventually becoming ineffective. The high dose of mAb and the possibility of immune complex formation may also reduce the efficacy of passive immunisation. An active vaccination strategy limits these complications.
Another approach to provide therapeutic agents for chronic asthma or other disease states with demonstrated eosinophilia or other conditions associated with IL-5 has been described in WO 97/45448. Therein, the use of “modified and variant forms of IL5 molecules capable of antagonising the activity of IL5” in ameliorating, abating or otherwise reducing the aberrant effects caused by native or mutant forms of IL5 has been proposed. The antagonizing effect is reported to be the result of the variant forms of IL5 binding to the low affinity a chain of IL5R but not to the high affinity receptors. By this way of action the variants compete with IL5 for binding to its receptors without exerting the physiological effects of IL5.
Eotaxin is a chemokine specific for Chemokine receptor 3, present on eosinophils, basophils and Th2 cells. However, Eotaxin has high specificity for eosinophils (Zimmerman et al., J. Immunol. 165: 5839–46 (2000)). Eosinophil migration is reduced by 70% in eotaxin-1 knock-out mice, which however can still develop eosinophilia (Rothenberg et al., J. Exp. Med. 185: 785–90 (1997)). IL-5 seems to be responsible for the migration of eosinophils from bone-marrow to blood, and eotaxin for the local migration in the tissue (Humbles et al., J. Exp. Med. 186: 601–12 (1997). Thus targeting eotaxin in addition to IL-5 may enhance immunotherapies directed towards lowering eosinophilia.
The human genome contains 3 eotaxin genes, eotaxin1–3 which share 30% homology. To date 2 genes are known in the mouse: eotaxin 1 and eotaxin 2 (Zimmerman et al., J. Immunol. 165: 5839–46 (2000)). They share 38% homology. Murine eotaxin-2 shares 59% homology with human eotaxin-2. In the mouse, eotaxin-1 seems to be ubiquitously expressed in the gastro-intestinal tract, while eotaxin-2 seems to be predominantly expressed in the jejunum (Zimmerman et al., J. Immunol. 165: 5839–46 (2000)). Eotaxin-1 is present in broncho-alveolar fluid (Teixeira et al., J. Clin. Invest. 100: 1657–66 (1997)). The sequence of human eotaxin-1 is shown in SEQ ID No.: 242 (aa 1–23 corresponds to the signal peptide), the sequence of human eotaxin-2 is shown in SEQ ID No.: 243 (aa 1–26 corresponds to the signal peptide), the sequence of human eotaxin-3 is shown in SEQ ID No.: 244 (aa 1–23 corresponds to the signal peptide), the sequence of mouse eotaxin-1 is shown in SEQ ID No.: 245 (aa 1–23 corresponds to the signal peptide), and the sequence of mouse eotaxin-2 is shown in SEQ ID No.: 246 (aa 1–23 corresponds to the signal peptide).
The monomer of eotaxin has a mass of 8.3 kDa and is in equilibrium with dimeric eotaxin over a wide range of conditions. The estimated Kd is 1.3 mM at 37° C. however the monomer is the predominant form (Crump et al., J. Biol. Chem. 273: 22471–9 (1998). The structure of Eotaxin has been elucidated by NMR spectroscopy. The binding site to its receptor CCR3, is at the N-terminus and the region preceding the first cysteine is crucial (Crump et al., J. Biol. Chem. 273: 22471–9 1998). Peptides derived from chemokine receptors bound to Eotaxin confirmed this finding. Eotaxin has four cysteines forming two disulfidebridges and can be chemically synthesized (Clark-Lewis et al., Biochemistry 30:3128–3135 1991). Eotaxin 1 is variably O-glcosylated on Thr 71(Noso, N. et al Eur J. Biochem. 253: 114–122). Expression of Eotaxin 1 in E. coli cytosol has also been described (Crump et al., J. Biol. Chem. 273: 22471–9 (1998)). Expression in E. coli as inclusion bodies with subsequent refolding (Mayer et al., Biochemistry 39: 8382–95 (2000)), and Insect cell expression (Forssmann et al., J. Exp. Med. 185: 2171–6 (1997)) have been reported for Eotaxin-2.
Interleukin 13 (IL-13) is secreted as a biologically active monomeric Th2 cytokine. The mature form of IL-13 comprises 112 amino acids in humans and 111 amino acids in mice. The calculated molecular mass of the protein is approximately 12.4 kDa. IL-13 can be N-linked glycosylated (Fitzgerald K. A. et al The Cytokines Fact Book 2nd edition Academic Press) IL-13 is produced by Th2 cells, mast cells, basophils and natural killer cells (Brombacher F, 2000 Bioessays July; 22(7):646–56). The functional IL-13 receptor is a heterodimer composed of the Interleukin 4 receptor α chain (IL-4R α chain) and one of the two IL-13 receptor α binding proteins (Brombacher F, 2000 Bioessays July; 22(7):646–56).
IL 13 plays a significant role in the pathology of asthma. It has been shown that IL 13 is involved in the central features of this disease. It has direct effects on allergen-induced airway hyperresponsiveness (AHR) and mucus production and has an involvement in eosinophilia (Kupenman D. A. 2002 Nature Medicine epub ahead of print). Selective neutralization of IL-13 in mice significantly attenuated the asthma phenotype. Furthermore, administration of IL-13 conferred an asthma-like phenotype to nonsensitized T-cell deficient or naive mice, respectively (Grünig G. et al., 1998 Science, 282(5397): 2261–3, Wills-Karp, M. et al, 1998 Science 282(5397): 2258–61). Mice with a targeted deletion of IL-13 failed to develop allergen-induced AHR and showed a marked decrease in mucus production (Walter, D. M. et al, 2001 J Immunol 167(8): 4668–75). Since IL-13 also influences eosinophilia in the murine asthma model (Grünig G. et al., 1998 Science, 282(5397): 2261–3), it possible IL-13 is involved in many more allergic diseases associated with eosinophilia and neutralizing its activity may offers a promising treatment for patients.
Additionally, upregulation of IL-13 and IL-13 receptor has been found in many tumor types (e.g. in all Hodgkin lymphoma disease cell lines examined to date). Thus immunization against IL-13 may provide a way of curing tumor patients overexpressing IL-13.
One way to improve the efficiency of vaccination is to increase the degree of repetitiveness of the antigen applied. Unlike isolated proteins, viruses induce prompt and efficient immune responses in the absence of any adjuvants both with and without T-cell help (Bachmann and Zinkernagel, Ann. Rev. Immunol: 15:235–270 (1991)). Although viruses often consist of few proteins, they are able to trigger much stronger immune responses than their isolated components. For B-cell responses, it is known that one crucial factor for the immunogenicity of viruses is the repetitiveness and order of surface epitopes. Many viruses exhibit a quasi-crystalline surface that displays a regular array of epitopes which efficiently crosslinks epitope-specific immunoglobulins on B cells (Bachmann and Zinkernagel, Immunol. Today 17:553–558 (1996)). This crosslinking of surface immunoglobulins on B cells is a strong activation signal that directly induces cell-cycle progression and the production of IgM antibodies. Further, such triggered B cells are able to activate T helper cells, which in turn induce a switch from IgM to IgG antibody production in B cells and the generation of long-lived B cell memory—the goal of any vaccination (Bachmann and Zinkernagel, Ann. Rev. Immunol. 15:235–270 (1997)). Viral structure is even linked to the generation of anti-antibodies in autoimmune disease and as a part of the natural response to pathogens (see Fehr, T., et al., J Exp. Med. 185:1785–1792 (1997)). Thus, antibodies presented by a highly organized viral surface are able to induce strong anti-antibody responses.
As indicated, however, the immune system usually fails to produce antibodies against self-derived structures. For soluble antigens present at low concentrations, this is due to tolerance at the Th cell level. Under these conditions, coupling the self-antigen to a carrier that can deliver T help may break tolerance. For soluble proteins present at high concentrations or membrane proteins at low concentration, B and Th cells may be tolerant. However, B cell tolerance may be reversible (anergy) and can be broken by administration of the antigen in a highly organized fashion coupled to a foreign carrier (Bachmann and Zinkernagel, Ann. Rev. Immunol. 15:235–270 (1997)).